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Abstract Background In screening of libraries derived by expression cloning, expression of active proteins in E. coli can be limited by formation of inclusion bodies. In these cases it would be desirable to enrich gene libraries for coding sequences with soluble gene products in E. coli and thus to improve the efficiency of screening. Previously Wilkinson and Harrison showed that solubility can be predicted from amino acid composition (Biotechnology 1991, 9(5):443–448). We have applied this analysis to members of the alpha/beta hydrolase fold family to predict their solubility in E. coli. alpha/beta hydrolases are a highly diverse family with more than 1800 proteins which have been grouped into homologous families and superfamilies. Results The predicted solubility in E. coli depends on hydrolase size, phylogenetic origin of the host organism, the homologous family and the superfamily, to which the hydrolase belongs. In general small hydrolases are predicted to be more soluble than large hydrolases, and eukaryotic hydrolases are predicted to be less soluble in E. coli than prokaryotic ones. However, combining phylogenetic origin and size leads to more complex conclusions. Hydrolases from prokaryotic, fungal and metazoan origin are predicted to be most soluble if they are of small, medium and large size, respectively. We observed large variations of predicted solubility between hydrolases from different homologous families and from different taxa. Conclusion A comprehensive analysis of all alpha/beta hydrolase sequences allows more efficient screenings for new soluble alpha/beta hydrolases by the use of libraries which contain more soluble gene products. Screening of hydrolases from families whose members are hard to express as soluble proteins in E. coli should first be done in coding sequences of organisms from phylogenetic groups with the highest average of predicted solubility for proteins of this family. The tools developed here can be used to identify attractive target genes for expression using protein sequences published in databases. This analysis also directs the design of degenerate, family- specific primers to amplify new members from homologous families or superfamilies with a high probability of soluble alpha/beta hydrolases.

Current diagnostic methods for Johne's disease in cattle allow reliable detection of infections with Mycobacterium avium ssp. paratuberculosis (MAP) not before animals are 2 years of age. Applying a flow cytometry-based approach (FCA) to quantify a MAP-specific interferon-gamma (IFN-γ) response in T cell subsets, the present study sought to monitor the kinetics of the cell-mediated immune response in experimentally infected calves. Six MAP-negative calves and six calves, orally inoculated with MAP at 10 days of age, were sampled every 4 weeks for 52 weeks post-inoculation (wpi). Peripheral blood mononuclear cells (PBMC) were stimulated with either purified protein derivatives (PPD) or whole cell sonicates derived from MAP (WCSj), M. avium ssp. avium or M. phlei for 6 days followed by labeling of intracellular IFN-γ in CD4 + and CD8 + T cells. No antigen-specific IFN-γ production was detectable in CD8 + cells throughout and the responses of CD4 + cells of MAP-infected and control calves were similar up to 12 wpi. However, the mean fluorescence intensity (MFI) for the detection of IFN-γ in CD4 + cells after WCSj antigen stimulation allowed for a differentiation of animal groups from 16 wpi onwards. This approach had a superior sensitivity (87.8%) and specificity (86.8%) to detect infected animals from 16 wpi onwards, i.e., in an early infection stage, as compared to the IFN-γ release assay (IGRA). Quantification of specific IFN-γ production at the level of individual CD4 + cells may serve, therefore, as a valuable tool to identify MAP-infected juvenile cattle., (© 2024. The Author(s).)

Antimicrobial resistance (AMR) is considered one of the greatest threats to both human and animal health. Efforts to address AMR include implementing antimicrobial stewardship programs and introducing alternative treatment options. Nevertheless, effective treatment of infectious diseases caused by bacteria will still require the identification and development of new antimicrobial agents. Eight different natural products were tested for antimicrobial activity against seven pathogenic bacterial species ( Brachyspira sp., Chlamydia sp., Clostridioides sp., Mannheimia sp., Mycobacterium sp., Mycoplasma sp., Pasteurella sp.). In a first pre-screening, most compounds (five out of eight) inhibited bacterial growth only at high concentrations, but three natural products (celastramycin A [CA], closthioamide [CT], maduranic acid [MA]) displayed activity at concentrations <2 µg/mL against Pasteurella sp. and two of them (CA and CT) also against Mannheimia sp. Those results were confirmed by testing a larger collection of isolates encompassing 64 Pasteurella and 56 Mannheimia field isolates originating from pigs or cattle, which yielded MIC 90 values of 0.5, 0.5, and 2 µg/mL against Pasteurella and 0.5, 4, and >16 µg/mL against Mannheimia for CA, CT, and MA, respectively. CA, CT, and MA exhibited higher MIC 50 and MIC 90 values against Pasteurella isolates with a known AMR phenotype against commonly used therapeutic antimicrobial agents than against isolates with unknown AMR profiles. This study demonstrates the importance of whole-cell antibacterial screening of natural products to identify promising scaffolds with broad- or narrow-spectrum antimicrobial activity against important Gram-negative veterinary pathogens with zoonotic potential.

Tuberculosis caused by Mycobacterium tuberculosis is a leading cause of death globally and a major health concern. In humans, macrophages are the first line invaded by M. tuberculosis. Upon infection, macrophages upregulate cyclooxygenase-2 (COX-2) expression and consequently elevate the formation of PGs, including PGE2 and PGD2. Although the role of proinflammatory PGE2 in M. tuberculosis infection has been reported, the roles of PGJ2 and 15-deoxy-PGJ2 (collectively named J2-PGs), the metabolites of PGD2 with anti-inflammatory features, remain elusive. In this study, we show that M. tuberculosis (H37Rv strain)-conditioned medium stimulates human monocyte-derived macrophages (MDMs) to elevate COX-2 expression along with robust generation of PGJ2, exceeding PGD2 formation, and to a minor extent also of 15-deoxy-PGJ2. Of interest, in M1-MDM phenotypes, PGJ2 and 15-deoxy-PGJ2 decreased M. tuberculosis (H37Rv strain)-conditioned medium-induced COX-2 expression and related PG formation by a negative feedback loop. Moreover, these J2-PGs downregulated the expression of the proinflammatory cytokines IL-6, IL-1β, and IFN-γ, but elevated the anti-inflammatory cytokine IL-10 and the M2 markers arginase-1 and CD163. These anti-inflammatory effects of J2-PGs in M1-MDM correlated with impaired activation of TGF-β-activated kinase 1/NF-κB/MAPK pathways. Finally, we found that J2-PGs regulate COX-2 expression, at least partially, via PGD2 receptor (DP1) and chemoattractant receptor homologue expressed on Th2 cells/DP2 receptors, but independent of the J2-PG receptor peroxisome proliferator-activated receptor-γ. Together, our findings reveal that M. tuberculosis induces COX-2 expression in human M1-MDMs, along with robust formation of J2-PGs that mediates anti-inflammatory effects via a negative feedback loop., (Copyright © 2023 by The American Association of Immunologists, Inc.)

Mycobacteria produce several unusual cofactors that contribute to their metabolic versatility and capability to survive in different environments. Mycofactocin (MFT) is a redox cofactor involved in ethanol metabolism. The redox-active core moiety of mycofactocin is derived from the short precursor peptide MftA, which is modified by several maturases. Recently, it has been shown that the core moiety is decorated by a β-1,4-glucan chain. Remarkably, the second glucose moiety of the oligosaccharide chain was found to be 2- O -methylated in Mycolicibacterium smegmatis . The biosynthetic gene responsible for this methylation, however, remained elusive, and no methyltransferase gene was part of the MFT biosynthetic gene cluster. Here, we applied reverse genetics to identify the gene product of MSMEG&#95;6237 ( mftM ) as the SAM-dependent methyltransferase was responsible for methylation of the cofactor in M. smegmatis . According to metabolic analysis and comparative genomics, the occurrence of methylated MFT species was correlated with the presence of mftM homologues in the genomes of mycofactocin producers. This study revealed that the pathogen Mycobacterium tuberculosis does not methylate mycofactocins. Interestingly, mftM homologues co-occur with both mycofactocin biosynthesis genes as well as the putative mycofactocin-dependent alcohol dehydrogenase Mdo. We further showed that mftM knock-out mutants of M. smegmatis suffer from a prolonged lag phase when grown on ethanol as a carbon source. In addition, in vitro digestion of the glucose chain by cellulase suggested a protective function of glucan methylation. These results close an important knowledge gap and provide a basis for future studies into the physiological functions of this unusual cofactor modification.

The Common Eider ( Somateria mollissima ) inhabits the entire northern hemisphere. In northern Europe, the flyway population reaches from the southern Wadden Sea to the northern Baltic coast. The European population is classified as endangered due to declines in Common Eider numbers across Europe since 1990. In this study, we assessed 121 carcasses of Common Eiders, captured incidentally in gillnets in the Western Baltic between 2017 and 2019. The most common findings were parasitic infections of the intestine by acanthocephalans in 95 animals, which correlated with enteritis in 50% of the cases. Parasites were identified as Profilicollis botulus in 25 selected animals. Additionally, oesophageal pustules, erosions, and ulcerations, presumably of traumatic origin, were frequently observed. Nephritis and hepatitis were frequent, but could not be attributed to specific causes. Lung oedema, fractures and subcutaneous haemorrhages likely resulted from entangling and drowning. Two Common Eiders had mycobacterial infections and in one of these, Mycobacterium avium subspecies (ssp.) avium was identified. This study gives an overview of morphological changes and infectious diseases from one location of the European flyway population. It contributes to future health studies on Common Eiders in the Baltic and Wadden Seas by providing baseline information to compare with other areas or circumstances.

Macrophages are the primary human host cells of intracellular Mycobacterium tuberculosis ( M.tb ) infection, where the magnitude of inflammatory reactions is crucial for determining the outcome of infection. Previously, we showed that the anti-inflammatory drug sulfasalazine (SASP) significantly reduced the M.tb bactericidal burden and histopathological inflammation in mice. Here, we asked which genes in human inflammatory macrophages are affected upon infection with M.tb and how would potential changes impact the functional state of macrophages. We used a flow cytometry sorting system which can distinguish the dead and alive states of M.tb harbored in human monocyte-derived macrophages (MDM). We found that the expression of cyclooxygenase-2 and microsomal prostaglandin E 2 synthase (mPGES)-1 increased significantly in tagRFP + MDM which were infected with alive M.tb . After exposure of polarized M1-MDM to M.tb (H37Rv strain)-conditioned medium (MTB-CM) or to the M.tb -derived 19-kD antigen, the production of PGE 2 and pro-inflammatory cytokines increased 3- to 4-fold. Upon treatment of M1-MDM with SASP, the MTB-CM-induced expression of COX-2 and the release of COX products and cytokines decreased. Elevation of PGE 2 in M1-MDM upon MTB-CM stimulation and modulation by SASP correlated with the activation of the NF-κB pathway. Together, infection of human macrophages by M.tb strongly induces COX-2 and mPGES-1 expression along with massive PGE 2 formation which is abrogated by the anti-inflammatory drug SASP., Competing Interests: The authors declare that the research presented was conducted in the absence of commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wang, Ning, Wang, Deng, Pace, Barth, Menge, Zhang, Dai, Cai, Chen and Werz.)

Bovine tuberculosis (bTB) not only poses a zoonotic threat to humans but also has a significant economic impact on livestock production in many areas of the world. Effective vaccines for humans, livestock, and wildlife are highly desirable to control tuberculosis. Suitable large animal models are indispensable for meaningful assessment of vaccine candidates. Here, we describe the refinement of an animal model for bTB in goats. Intrabronchial inoculation procedure via video-guided endoscopy in anesthetized animals, collection of lungs after intratracheal fixation in situ , and imaging of lungs by computed tomography (CT) were established in three goats using barium sulfate as surrogate inoculum. For subsequent infection experiments, four goats were infected with 4.7 × 10 2 colony-forming units of M. bovis by intrabronchial inoculation using video-guided endoscopy with spray catheters. Defined amounts of inoculum were deposited at five sites per lung. Four age-matched goats were mock-inoculated. None of the goats developed clinical signs until they were euthanized 5 months post infection, but simultaneous skin testing confirmed bTB infection in all goats inoculated with M. bovis . In tissues collected at necropsy, M. bovis was consistently re-isolated from granulomas in lymph nodes, draining the lungs of all the goats infected with M. bovis . Further dissemination was observed in one goat only. Pulmonary lesions were quantified by CT and digital 2D radiography (DR). CT revealed mineralized lesions in all the infected goats ranging from <5 mm to >10 mm in diameter. Small lesions <5 mm predominated. The DR failed to detect small lesions and to determine the exact location of lesions because of overlapping of pulmonary lobes. Relative volume of pulmonary lesions was low in three but high in one goat that also had extensive cavitation. CT lesions could be correlated to gross pathologic findings and histologic granuloma types in representative pulmonary lobes. In conclusion, video-guided intrabronchial inoculation with spray catheters, mimicking the natural way of infection, resulted in pulmonary infection of goats with M. bovis . CT, but not DR, presented as a highly sensitive method to quantify the extent of pulmonary lesions. This goat model of TB may serve as a model for testing TB vaccine efficacy., Competing Interests: LG was employed by Vakzine Projekt Management GmbH. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wedlich, Figl, Liebler-Tenorio, Köhler, von Pückler, Rissmann, Petow, Barth, Reinhold, Ulrich, Grode, Kaufmann and Menge.)

Bovine tuberculosis (bTB) caused by Mycobacterium (M.) bovis and M. caprae is a transmissible disease of livestock, notifiable to the World Organization for Animal Health (OIE). BTB particularly affects cattle and small ruminants and can be transmitted to humans thereby posing a significant threat to veterinary and public health worldwide. M. bovis is the principal cause of bTB in Algeria. In order to better understand the route of spreading and elaborate an eradication program, isolation and characterization of mycobacteria from Algerian cattle was performed. Sixty strains belonging to the M. tuberculosis complex were analyzed by spoligotyping, thereof 42 by 19-locus-MIRU-VNTR-typing. Spoligotyping revealed 16 distinguishable patterns (Hunter-Gaston discriminatory index [HGDI] of 0.8294), with types SB0120 (n = 20) and SB0121 (n = 13) being the most frequent patterns, representing 55% of the strains. Analyses based on 19-locus-MIRU-VNTR yielded 32 different profiles, five clusters and one orphan pattern, showing higher discriminatory power (HGDI = 0.9779) than spoligotyping. Seven VNTR-loci [VNTR 577 (alias ETR C), 2163b (QU11b), 2165 (ETR A), 2461 (ETR B), 3007 (MIRU 27), 2163a (QUB11a) and 3232 (QUB 3232)] were the most discriminative loci (HGDI ˃ 0.50). In conclusion, 19-locus-MIRU-VNTR yielded more information than spoligotyping concerning molecular differentiation of strains and better supports the elucidation of transmission routes of M. bovis between Algerian cattle herds., Competing Interests: The authors have declared that no competing interests exist.

Salmonella enterica ssp. enterica serovars Enteritidis (SE) and Gallinarum (SG) cause different diseases in chickens. However, both are able to reach the blood stream where heterophils and monocytes are potentially able to phagocytose and kill the pathogens. Using an ex vivo chicken whole blood infection model, we compared the complex interactions of the differentially host-adapted SE and SG with immune cells in blood samples of two White Leghorn chicken lines showing different laying performance (WLA: high producer; R11: low producer). In order to examine the dynamic interaction between peripheral blood leucocytes and the Salmonella serovars, we performed flow cytometric analyses and survival assays measuring (i) leucocyte numbers, (ii) pathogen association with immune cells, (iii) Salmonella viability and (iv) immune gene transcription in infected whole blood over a four-hour co-culture period. Inoculation of blood from the two chicken lines with Salmonella led primarily to an interaction of the bacteria with monocytes, followed by heterophils and thrombocytes. We found higher proportions of monocytes associated with SE than with SG. In blood samples of high producing chickens, a decrease in the numbers of both heterophils and Salmonella was observed. The Salmonella challenge induced transcription of interleukin-8 (IL-8) which was more pronounced in SG- than SE-inoculated blood of R11. In conclusion, the stronger interaction of monocytes with SE than SG and the better survivability of Salmonella in blood of low-producer chickens shows that the host-pathogen interaction and the strength of the immune defence depend on both the Salmonella serovar and the chicken line., (© 2021. The Author(s).)

Tapirs are a taxonomic group with a high susceptibility to mycobacterial diseases. However, successful therapy has only been documented sporadically. Here treatment of mycobacteriosis diagnosed in three, one male and two female, lowland tapirs ( Tapirus terrestris ) in a zoo in Germany is reported. Two of the animals showed chronic mild respiratory signs, and conventional therapy did not improve the condition. Culture of broncho-alveolar lavage (BAL) samples was positive for Mycobacterium avium ssp. hominissuis . Upon airway endoscopy, bronchial edema and increased mucus production were visible. Initially, all three infected tapirs received oral antimycobacterial therapy consisting of 5 mg/kg body weight isoniazid, 10 mg/kg rifampicin, and 10 mg/kg clarithromycin q24h. Based on therapeutic drug level monitoring, the doses of rifampicin were adjusted to 12 and 15 mg/kg in the females and the male, respectively. The treatment with all three drugs was continued for 11 mon. Six months into treatment, the clinical condition resolved, and repeated BAL samples of all three tapirs tested negative for mycobacteria by culture. Here the approach for a treatment protocol with minimal side effects suitable to control infections with nontuberculous mycobacteria in lowland tapirs is reported.

Tapirs seem particularly susceptible to mycobacterial infections, especially to tuberculosis caused by M. tuberculosis or M. bovis. In this case series, we report an infection with the non-tuberculous mycobacteria (NTM) species M. avium ssp. hominissuis (MAH) in a group of four (2.2) captive lowland tapirs (Tapirus terrestris). Two female tapirs showed mild respiratory signs such as coughing and mucous sputum production for several years, one juvenile male tapir had to be euthanized due to severe dyspnoea, and the adult male only showed mild respiratory signs in 2010. Post-mortem histopathology of the euthanized animal revealed a chronic bronchopneumonia, and MAH was detected via culture. Subsequently, the three remaining tapirs were tested further: serologically, the tapirs had high antibody titres against M. avium, but they showed no reaction in the comparative skin test (TST). At several time points, the animals were tested for the presence of mycobacteria in different sample matrices including sputum samples, pooled faecal samples as well as swabs from the tapir enclosure to identify potential environmental niches of the pathogen. Moreover, animals were directly sampled using nasal swabs, endoscopic broncho-alveolar (BAL) and gastric lavages. MAH was detected by culture in the sputum samples, in the BAL of the breeding pair, as well as in the swimming pool water and walls, and in swabs taken from the tapir's sleeping beds. We conclude that the TST is not a useful diagnostic tool to detect MAC infections in tapirs, whereas antibody ELISA and culture from BAL appear more sensitive., (© 2020 The Authors. Transboundary and Emerging Diseases published by Wiley-VCH GmbH.)

Cattle and other ruminants are primary reservoirs for Shiga toxin-producing Escherichia coli (STEC) strains which have a highly variable, but unpredictable, pathogenic potential for humans. Domestic swine can carry and shed STEC, but only STEC strains producing the Shiga toxin (Stx) 2e variant and causing edema disease in piglets are considered pathogens of veterinary medical interest. In this chapter, we present general diagnostic workflows for sampling livestock animals to assess STEC prevalence, magnitude, and duration of host colonization. This is followed by detailed method protocols for STEC detection and typing at genetic and phenotypic levels to assess the relative virulence exerted by the strains.

Mycobacteriosis has been rarely described in pet rabbits (Oryctolagus cuniculus). Here we present two cases of intestinal mycobacteriosis from north-eastern Germany. The first adult rabbit was euthanized due to severe cardiovascular failure, hypothermia and chronic weight loss. Necropsy revealed cachexia and a focal, fibrinonecrotic lesion in the caecum. Histologically, severe granulomatous inflammation, with numerous multinucleated giant cells and abundant acid-fast bacilli, was detected under the fibrinonecrotic material in the abdominal wall adjacent to the caecal lesion, caecal lymph nodes, spleen, liver and lungs. Microbiological culture detected Mycobacterium avium subspecies hominissuis, Escherichia coli, Clostridium disporicum and Bacteroides ovatus. A retrospective assessment of 2,013 other pet rabbit necropsies, performed between 1995 and 2019, revealed one additional case of intestinal mycobacteriosis. This animal had been euthanized due to persistent hindlimb lameness and necropsy revealed comminuted fractures of the pelvic bones and multiple large liquefied abscess-like lesions in the caecal and colonic walls. Histology revealed granulomatous inflammation with acid-fast bacilli. Polymerase chain reaction on formalin-fixed, paraffin-embedded tissue identified the presence of M. avium spp. In contrast to European wild rabbits (Oryctolagus cuniculus) from Scotland, these findings indicate that intestinal mycobacteriosis is rare in pet rabbits from north-eastern Germany. Zoonotic potential should be considered due to the close contact between pets and their owners and the chronic course of the disease with an initial lack of clinical signs., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Cattle harbor Shiga toxin-producing Escherichia coli (STEC) in their intestinal tract, thereby providing these microorganisms with an ecological niche, but without this colonization leading to any clinical signs. In a preceding study, genotypic characterization of bovine STEC isolates unveiled that their ability to colonize cattle persistently (STEC per ) or only sporadically (STEC spo ) is more closely associated with the overall composition of the accessory rather than the core genome. However, the colonization pattern could not be unequivocally linked to the possession of classical virulence genes. This study aimed at assessing, therefore, if the presence of certain phenotypic traits in the strains determines their colonization pattern and if these can be traced back to distinctive genetic features. STEC spo strains produced significantly more biofilm than STEC per when incubated at lower temperatures. Key substrates, the metabolism of which showed a significant association with colonization type, were glyoxylic acid and L-rhamnose, which were utilized by STEC spo , but not or only by some STEC per . Genomic sequences of the respective glc and rha operons contained mutations and frameshifts in uptake and/or regulatory genes, particularly in STEC per . These findings suggest that STEC spo conserved features leveraging survival in the environment, whereas the acquisition of a persistent colonization phenotype in the cattle reservoir was accompanied by the loss of metabolic properties and genomic mutations in the underlying genetic pathways.

A 9-month old pet ferret was presented to a veterinarian with symptoms of weight loss, apathy, and hyporexia. Explorative laparotomy identified a firm mass of approximately 2 × 2 × 2 cm in size in the mesentery of the jejunum. Because of the poor general condition and the unfavorable prognosis, the ferret was euthanized during surgery. The mass was resected in total and submitted to histological examination which revealed a granulomatous and necrotizing lymphadenitis. Acid fast bacteria were detected by Fite-Faraco staining leading to the suspicion of an infection with Mycobacteria sp. PCR confirmed presence of DNA of members of the Mycobacterium tuberculosis complex, subsequently specified as M. bovis . The detected spoligotype SB2548 was described for the first time. Ferrets are presented to veterinarians with increasing frequency because of their growing popularity as pet animals. Since these animals are highly susceptible to mycobacterial infections, mycobacteriosis and especially zoonotic relevant tuberculosis should be considered as differential diagnosis., Competing Interests: The authors confirm that they do not have any conflict of interest., (© Georg Thieme Verlag KG Stuttgart · New York.)

The objective of our study was to provide a molecular analysis using DNA-microarray based assays of commensal E. coli populations from apparently healthy livestock and their attendants to assess the virulence potential as well as multidrug resistance (MDR) genotypes. We randomly collected 132 fecal samples from seemingly healthy smallholder´s food producing animals [buffalo (n = 32) and cattle (n = 50)] as well as from contacting farmers (n = 50). Bacterial isolation and identification were performed using standard protocols, while E. coli isolates were characterized using a DNA microarray system targeting 60 different virulence and 47 antibiotic resistance genes of clinical importance and allowing assignment to most common H and O types. From the fecal samples examined, 47 E. coli isolates were obtained. The array predicted serotypes for 14 out of the 47 E. coli isolates. Six E. coli isolates were identified as STEC since Shiga toxin genes were detected. In summary, 36 different virulence genes were identified; of which, hemL, lpfA and iss were most prevalent. Thirty-four E. coli isolates were found to carry at least one antimicrobial resistance gene. Of these, 20 did exhibit genes allowing strain classification as MDR. More than half of the isolates contained antimicrobial resistance genes associated with beta lactam resistance 27/47 (57.5 %). The 13 remaining isolates did not contain any resistance gene tested with the array. Our study demonstrated the presence of antimicrobial resistance genes and virulence genotypes among commensal E. coli of human and animal sources., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Mycobacterium bovis is the main agent of bovine tuberculosis, but has also zoonotic potential. An 8-month-old female domestic shorthaired cat imported from Ukraine developed wound complications after abdominal surgery. A second surgery performed in Germany showed a focal, partly cystic mass within the mesentery. Despite antimicrobial treatment, the cat did not recover and was humanely destroyed. Grossly, several abdominal lymph nodes were enlarged. Histopathology revealed a mild to moderate, multifocal, granulomatous to pyogranulomatous, partially necrotizing inflammation, most prominent in the abdominal cavity. Within the lesions there were acid-fast bacilli within the cytoplasm of macrophages demonstrated by Ziehl-Neelsen staining. Further investigations revealed M. bovis SB0950 in the affected tissues., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Tuberculosis (TB) occurs in a wide range of mammalian species and thus poses a health risk to humans living or working in close proximity with TB infected animals. Despite a high incidence of M. bovis infections in domestic or wildlife species tuberculosis infections in rhinoceros have so far been very limited. Over the past 53 years, tuberculosis of the respiratory tract has been confirmed in just 22 rhinoceros, most of those infected not by M. bovis but M. tuberculosis. However, because of the zoonotic risk TB testing is recommended or becomes even mandatory in endangered species. The dilemma in rhinoceros and many other wildlife species; non-validated tests are highly inconsistent in their ability to identify TB infection. Current lack of TB diagnostics may result in TB positive rhinoceros living with the infection, transmitting it to those around them or in euthanasia of animals found unconfirmed at necropsy. This is an unacceptable diagnostic status considering that some species are critically endangered and therefore should not be euthanized in order to confirm suspicion of disease. To overcome this shortcoming we used bronchoscopy to detect mycobacteria in respiratory fluids of TB suspicious rhinoceros. Fluids from seven, TB suspicious white rhinoceros were harvested during 21 bronchoscopies. Our new approach: In addition to bacterial culture a dual quantitative PCR system tested for the general presence of DNA from NTM and more specifically for DNA from MTC. Both, bacterial culture and qPCR were negative for MTC in respiratory fluids of all rhinoceros (7/7). At the same time, respiratory fluids from six rhinoceros tested positive for the presence of NTM or other closely related bacteria (6/7). M. tuberculosis was found only once in an oesophageal aspirate. The high incidence of mycobacterial DNA in the respiratory tract suggests that white rhinoceros, as strict grazers, are immensely exposed to environmental bacteria of this genus. Presence of NTM in the respiratory or intestinal system could possibly cause false positive results in intradermal tests. A wider use of bronchoalveolar lavage is warranted to further elucidate immunologic response to NTM and exposure to, incidence and prevalence of MTC infections in rhinoceros., Competing Interests: Commercial affiliations of authors to Conservation Medicine Services [JC] and the Garston Veterinary Group [DW] do not alter the adherence to all PLOS ONE policies on sharing data and materials. There are no patents, products in development or marketed products to declare.

Vitamin E (vit E), an essential antioxidant for maintaining the stability of biological membranes and the function of the immune system, is considered to support adaptive immune responses and performance in cattle. The principal virulence factor of Shiga toxin (Stx)-producing Escherichia coli (STEC), the eponymous Stx, modulates cellular immune responses in cattle, the primary STEC reservoir. Active and passive immunization of calves with Shiga toxoids (rStx MUT ) was recently shown to reduce the STEC shedding. Here, we examined the influence of vit E on calves' serum α-tocopherol, performance, haematology, blood chemistry and its interaction with rStx MUT immunization. Data from calves having received passive (colostrum from immunized cows) and active (intramuscularly at 5th and 8th weeks of life) vaccination with rStx MUT (n = 24) were compared to unvaccinated controls (n = 24; fed with low anti-Stx colostrum, placebo injected). For each vaccination group, data were analysed according to the level of vit E supplementation offered by milk replacer (188 IU all-rac-α-tocopheryl acetate daily [VitE M ] vs. 354 IU [VitE H ]). An increase by 79% in daily vit E supplementation led to slightly higher serum α-tocopherol level and earlier concentrate intake at the beginning of the experiment without significant differences in live weight gain, haematology, blood chemistry parameters and peripheral CD4 + and CD8 + T-cell subpopulations. rStx MUT vaccination modulated the CD4 + /CD8 + ratio irrespective of vit E supplementation but decreased concentrate intake in VitE H in a time-dependent manner. Results of our study indicate that an increase in daily vit E supplementation vastly fails to exert effects on laboratory parameters and growth performance. However, observed interactive effects of vit E supply and vaccination on the regulation of feed intake deserves further attention., (© 2018 Blackwell Verlag GmbH.)

In 2011, Germany was struck by the largest outbreak of hemolytic uremic syndrome. The highly virulent E. coli O104:H4 outbreak strain LB226692 possesses a blended virulence profile combining genetic patterns of human adapted enteroaggregative E. coli (EAEC), rarely detected in animal hosts before, and enterohemorrhagic E. coli (EHEC), a subpopulation of Shiga toxin (Stx)-producing E. coli (STEC) basically adapted to the ruminant host. This study aimed at appraising the relative level of adaptation of the EAEC/EHEC hybrid strain LB226692 to humans and cattle. Adherence and invasion of the hybrid strain to intestinal (jejunal and colonic) epithelial cells (IEC) of human and bovine origin was compared to that of E. coli strains representative of different pathovars and commensal E. coli by means of light and electron microscopy and culture. Strain-specific host gene transcription profiles of selected cytokines and chemokines as well as host-induced transcription of bacterial virulence genes were assessed. The release of Stx upon host cell contact was quantified. The outbreak strain's immunomodulation was assessed by cultivating primary bovine macrophages with conditioned supernatants from IEC infection studies with E. coli, serving as model for the innate immunity of the bovine gut. The outbreak strain adhered to IEC of both, human and bovine origin. Electron microscopy of infected cells revealed the strain's particular affinity to human small IEC, in contrast to few interactions with bovine small IEC. The outbreak strain possessed a high-level of adhesive power, similar to human-associated E. coli strains and in contrast to bovine-associated STEC strains. The outbreak strain displayed a non-invasive phenotype, in contrast to some bovine-associated E. coli strains, which were invasive. The outbreak strain provoked some pro-inflammatory activity in human cells, but to a lower extent as compared to other pathotypes. In contrasts to bovine-associated E. coli strains, the outbreak strain induced marked pro-inflammatory activity when interacting with bovine host cells directly (IEC) and indirectly (macrophages). Among stx2-positive strains, the human-pathogenic strains (LB226692 and EHEC strain 86-24) released higher amounts of Stx compared to bovine-associated STEC. The findings imply that the outbreak strain is rather adapted to humans than to cattle. However, the outbreak strain's potential to colonize IEC of both host species and the rather mixed reaction patterns observed for all strains under study indicate, that even STEC strains with an unusual genotype as the EHEC O104:H4 outbreak strain, i.e. with an EAEC genetic background, may be able to conquer other reservoir hosts., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Escherichia coli O157:H7 is a zoonotic pathogen of global importance and the serotype of Shiga toxin-producing E.coli (STEC) most frequently associated with Hemolytic Uremic Syndrome (HUS) in humans. The main STEC reservoir is cattle. Vaccination of calves with the carboxy-terminal fraction of Intimin γ (IntC280) and EspB can reduce E.coli O157:H7 fecal shedding after experimental challenge. Shiga toxin (Stx) exerts local immunosuppressive effects in the bovine intestine and Stx2B fused to Brucella lumazine synthase (BLS-Stx2B) induces Stx2-neutralizing antibodies. To determine if an immune response against Stx could improve a vaccine's effect on fecal shedding, groups of calves were immunized with EspB + IntC280, with EspB + IntC280 + BLS-Stx2B, or kept as controls. At 24 days post vaccination calves were challenged with E.coli O157:H7. Shedding of E.coli O157:H7 was assessed in recto-anal mucosal swabs by direct plating and enrichment followed by immunomagnetic separation and multiplex PCR. Calves were euthanized 15 days after the challenge and intestinal segments were obtained to assess mucosal antibodies. Vaccination induced a significant increase of IntC280 and EspB specific antibodies in serum and intestinal mucosa in both vaccinated groups. Antibodies against Stx2B were detected in serum and intestinal mucosa of animals vaccinated with 3 antigens. Sera and intestinal homogenates were able to neutralize Stx2 verocytotoxicity compared to the control and the 2-antigens vaccinated group. Both vaccines reduced E.coli O157:H7 shedding compared to the control group. The addition of Stx2B to the vaccine formulation did not result in a superior level of protection compared to the one conferred by IntC280 and EspB alone. It remains to be determined if the inclusion of Stx2B in the vaccine alters E.coli O157:H7 shedding patterns in the long term and after recurrent low dose exposure as occurring in cattle herds., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

On the Mediterranean island of Corsica, cohabitation between sympatric domestic pigs and Eurasian wild boar (Sus scrofa) is common and widespread and can facilitate the maintenance and dissemination of several pathogens detrimental for the pig industry or human health. In this study, we monitored a population of free-ranging domestic pigs reared in extensive conditions within a 800-ha property located in Central Corsica which was frequently visited by a sympatric population of wild boar between 2013 and 2015. We used GPS collars to assess evidence of a spatially shared environment. Subsequently, we analysed by PFGE of XbaI-restricted DNA if those populations shared faecal Escherichia coli clones that would indicate contact and compared these results with those collected in a distant (separated by at least 50 km) population of wild boar used as control. Results showed that one of eight wild boars sampled in the study area shed E. coli XbaI clones identical to clones isolated from domestic pig sounders from the farm, while wild boar populations sampled in distant parts of the study area shared no identical clone with the domestic pigs monitored. Interestingly, within the sampled pigs, two identical clones were found in 2013 and in 2015, indicating a long-time persisting colonization type. Although the method of isolation of E. coli and PFGE typing of the isolates requires intensive laboratory work, it is applicable under field conditions to monitor potential infectious contacts. It also provides evidence of exchange of microorganisms between sympatric domestic pigs and wild boar populations., (© 2018 Blackwell Verlag GmbH.)

The principal virulence factor of Shiga toxin (Stx)-producing Escherichia coli (STEC), the eponymous Stx, modulates cellular immune responses in cattle, the primary STEC reservoir. We examined whether immunization with genetically inactivated recombinant Shiga toxoids (rStx1 MUT /rStx2 MUT ) influences STEC shedding in a calf cohort. A group of 24 calves was passively (colostrum from immunized cows) and actively (intra-muscularly at 5 th and 8 th week) vaccinated. Twenty-four calves served as unvaccinated controls (fed with low anti-Stx colostrum, placebo injected). Each group was divided according to the vitamin E concentration they received by milk replacer (moderate and high supplemented). The effective transfer of Stx-neutralizing antibodies from dams to calves via colostrum was confirmed by Vero cell assay. Serum antibody titers in calves differed significantly between the vaccinated and the control group until the 16 th week of life. Using the expression of activation marker CD25 on CD4 + CD45RO + cells and CD8α hi CD45RO + cells as flow cytometry based read-out, cells from vaccinated animals responded more pronounced than those of control calves to lysates of STEC and E. coli strains isolated from the farm as well as to rStx2 MUT in the 16 th week. Summarized for the entire observation period, less fecal samples from vaccinated calves were stx 1 and/or stx 2 positive than samples from control animals when calves were fed a moderate amount of vitamin E. This study provides first evidence, that transfer to and induction in young calves of Stx-neutralizing antibodies by Shiga toxoid vaccination offers the opportunity to reduce the incidence of stx-positive fecal samples in a calf cohort.

Swine dysentery (SD) is an economically important diarrheal disease in pigs caused by different strongly hemolytic Brachyspira (B.) species, such as B. hyodysenteriae, B. suanatina and B. hampsonii. Possible associations of epidemiologic data, such as multilocus sequence types (STs) to virulence gene profiles and antimicrobial susceptibility are rather scarce, particularly for B. hyodysenteriae isolates from Germany. In this study, B. hyodysenteriae (n = 116) isolated from diarrheic pigs between 1990 and 2016 in Germany were investigated for their STs, susceptibility to the major drugs used for treatment of SD (tiamulin and valnemulin) and genes that were previously linked with virulence and encode for hemolysins (tlyA, tlyB, tlyC, hlyA, BHWA1&#95;RS02885, BHWA1&#95;RS09085, BHWA1&#95;RS04705, and BHWA1&#95;RS02195), outer membrane proteins (OMPs) (bhlp16, bhlp17.6, bhlp29.7, bhmp39f, and bhmp39h) as well as iron acquisition factors (ftnA and bitC). Multilocus sequence typing (MLST) revealed that 79.4% of the isolates belonged to only three STs, namely ST52 (41.4%), ST8 (12.1%), and ST112 (25.9%) which have been observed in other European countries before. Another 24 isolates belonged to twelve new STs (ST113-118, ST120-123, ST131, and ST193). The temporal distribution of STs revealed the presence of new STs as well as the regular presence of ST52 over three decades (1990s-2000s). The proportion of strains that showed resistance to both tiamulin und valnemulin (39.1%) varied considerably among the most frequent STs ranging from 0% (0/14 isolates resistant) in ST8 isolates to 46.7% (14/30), 52.1% (25/48), and 85.7% (6/7) in isolates belonging to ST112, ST52, and ST114, respectively. All hemolysin genes as well as the iron-related gene ftnA and the OMP gene bhlp29.7 were regularly present in the isolates, while the OMP genes bhlp17.6 and bhmp39h could not be detected. Sequence analysis of hemolysin genes of selected isolates revealed co-evolution of tlyB, BHWA1&#95;RS02885, BHWA1&#95;RS09085, and BHWA1&#95;RS02195 with the core genome and suggested independent evolution of tlyA, tlyC, and hlyA. Our data indicate that in Germany, swine dysentery might be caused by a limited number of B. hyodysenteriae clonal groups. Major STs (ST8, ST52, and ST112) are shared with other countries in Europe suggesting a possible role of the European intra-Community trade of pigs in the dissemination of certain clones. The identification of several novel STs, some of which are single or double locus variants of ST52, may on the other hand hint towards an ongoing diversification of the pathogen in the studied area. The linkage of pleuromutilin susceptibility and sequence type of an isolate might reflect a clonal expansion of the underlying resistance mechanism, namely mutations in the ribosomal RNA genes. A linkage between single virulence-associated genes (VAGs) or even VAG patterns and the phylogenetic background of the isolates could not be established, since almost all VAGs were regularly present in the isolates.

We assessed the ability of a commercial DNA microarray to characterize bovine Shiga toxin-producing Escherichia coli (STEC) isolates and evaluated the results using in silico hybridization of the microarray probes within whole genome sequencing scaffolds. From a total of 69,954 reactions (393 probes with 178 isolates), 68,706 (98.2%) gave identical results by DNA microarray and in silico probe hybridization. Results were more congruent when detecting the genoserotype (209 differing results from 19,758 in total; 1.1%) or antimicrobial resistance genes (AMRGs; 141 of 26,878; 0.5%) than when detecting virulence-associated genes (VAGs; 876 of 22,072; 4.0%). Owing to the limited coverage of O-antigens by the microarray, only 37.2% of the isolates could be genoserotyped. However, the microarray proved suitable to rapidly screen bovine STEC strains for the occurrence of high numbers of VAGs and AMRGs and is suitable for molecular surveillance workflows.

Shiga toxin-producing Escherichia coli (STEC) strains are important zoonotic enteric pathogens with the main reservoir in cattle. Here, we present the genomes of two STEC strains and one atypical enteropathogenic E. coli strain from cattle origin, obtained during a longitudinal study in German cattle herds., (Copyright © 2017 Geue et al.)

Burkholderia mallei is a Gram-negative coccobacillus which causes glanders-a fatal disease of equines that may occasionally be transmitted to humans. Several cases of outbreaks have been reported from India since 2006. This paper presents draft genome sequences of two B. mallei strains isolated from equines affected by glanders in India., (Copyright © 2017 Singha et al.)

Brachyspira (B.) hyodysenteriae the causative agent of swine dysentery (SD) has been divided into 9 serotypes on basis of its lipooligosaccharide (LOS). Knowledge on circulating serotypes in Europe, however, is rare. Regarding that immunity to SD is serotype specific an update of B. hyodysenteriae serotyping was undertaken. A LOS band of 10 to 25kDa was identified being appropriate for this purpose. Isolates from Germany, Spain, Denmark, USA and Japan were characterized in the immunoblot by sera raised to serotypes 1 through 7, serogroups H and I (reference strains) and to eight German strains. In total, 57 (51%) isolates responded to at least one of the antisera. Regarding German isolates (n=75) only 35 (46.7%) were identified but mainly by antisera to German strains. Positive Spanish isolates (12 of 17) yielded similar results. In contrast, positively reacting Danish isolates (9 of 12) were mainly identified by antisera to the reference strains as it was the case for recent U.S. (1 of 8) and Japanese isolates (3 of 5). Results indicate that B. hyodysenteriae has a high degree of serological heterogeneity that has probably differently developed in diverse geographical areas over time. This situation represents a challenge for vaccine development., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Prevention of enterohemorrhagic Escherichia coli (EHEC) O157:H7 infections and of their severe clinical sequelae in humans remain to be a current challenge. Administration of bovine lactoferrin (bLF) proved to be effective in clearing EHEC from the bovine intestine, an important EHEC reservoir, suggesting that bLF may also be beneficial in human application against EHEC infections. To estimate the biological safety of this approach, we analyzed the effects of bLF on the main EHEC virulence factor, Shiga toxin (Stx). We quantified the release of Stx 1 and 2 from two O157:H7 EHEC strains (Stx1 + Stx2 + and Stx2 + producing, respectively) cultured in the presence of bLF using ELISA assays and assessed cytotoxic effects of bLF and co-cultured EHEC on Vero cells. Effects of bLF on the stability of Stx2 were investigated using western blotting. ELISA results indicate a bLF concentration-dependent decrease of active, cell-free Stx2, but not Stx1 in EHEC cultures. High concentrations (100 and 50mg/ml) of bLF resulted in significantly reduced (p<0.05) metabolic activity rates of Vero cells, whereas a concentration of 10mg/ml bLF was considered non-toxic for Vero cells. At concentrations of 1 or 0.1mg/ml, bLF mitigated the verocytotoxicity of EHEC strains in a co-culture model up to 48h after inoculation. When only colonizing bacteria were taken into account, cytotoxicity could be significantly reduced by 10 and 1mg/ml bLF during 48h. This effect of bLF at least partly results from degradation of the Stx2 receptor-binding B-subunit., (Copyright © 2016. Published by Elsevier B.V.)

Shiga toxin-producing Escherichia coli (STEC) comprise a group of zoonotic enteric pathogens with ruminants, especially cattle, as the main reservoir. O-antigens are instrumental for host colonization and bacterial niche adaptation. They are highly immunogenic and, therefore, targeted by the adaptive immune system. The O-antigen is one of the most diverse bacterial cell constituents and variation not only exists between different bacterial species, but also between individual isolates/strains within a single species. We recently identified STEC persistently infecting cattle and belonging to the different serotypes O156:H25 ( n = 21) and O182:H25 ( n = 15) that were of the MLST sequence types ST300 or ST688. These STs differ by a single nucleotide in purA only. Fitness-, virulence-associated genome regions, and CRISPR/CAS (clustered regularly interspaced short palindromic repeats/CRISPR associated sequence) arrays of these STEC O156:H25 and O182:H25 isolates were highly similar, and identical genomic integration sites for the stx converting bacteriophages and the core LEE, identical Shiga toxin converting bacteriophage genes for stx1a , identical complete LEE loci, and identical sets of chemotaxis and flagellar genes were identified. In contrast to this genomic similarity, the nucleotide sequences of the O-antigen gene cluster (O-AGC) regions between galF and gnd and very few flanking genes differed fundamentally and were specific for the respective serotype. Sporadic aEPEC O156:H8 isolates ( n = 5) were isolated in temporal and spatial proximity. While the O-AGC and the corresponding 5' and 3' flanking regions of these aEPEC isolates were identical to the respective region in the STEC O156:H25 isolates, the core genome, the virulence associated genome regions and the CRISPR/CAS elements differed profoundly. Our cumulative epidemiological and molecular data suggests a recent switch of the O-AGC between isolates with O156:H8 strains having served as DNA donors. Such O-antigen switches can affect the evaluation of a strain's pathogenic and virulence potential, suggesting that NGS methods might lead to a more reliable risk assessment.

In 2011, a severe outbreak of hemolytic-uremic syndrome was caused by an unusual, highly virulent enterohemorrhagic E. coli (EHEC) O104:H4 strain, which possessed EHEC virulence traits in the genetic background of human-adapted enteroaggregative E. coli. To determine magnitude of fecal shedding and site of colonization of EHEC O104:H4 in a livestock host, 30 (ten/strain) weaned calves were inoculated with 10(10) CFU of EHEC O104:H4, EHEC O157:H7 (positive control) or E. coli strain 123 (negative control) and necropsied (4 or 28 d.p.i.). E. coli O157:H7 was recovered until 28 d.p.i. and O104:H4 until 24 d.p.i. At 4 d.p.i., EHEC O104:H4 was isolated from intestinal content and detected associated with the intestinal mucosa. These results are the first evidence that cattle, the most important EHEC reservoir, can also carry unusual EHEC strains at least transiently, questioning our current understanding of the molecular basis of host adaptation of this important E. coli pathovar.

Unlabelled: Shiga toxin-producing Escherichia coli (STEC) strains can colonize cattle for several months and may, thus, serve as gene reservoirs for the genesis of highly virulent zoonotic enterohemorrhagic E. coli (EHEC). Attempts to reduce the human risk for acquiring EHEC infections should include strategies to control such STEC strains persisting in cattle. We therefore aimed to identify genetic patterns associated with the STEC colonization type in the bovine host. We included 88 persistent colonizing STEC (STEC(per)) (shedding for ≥4 months) and 74 sporadically colonizing STEC (STEC(spo)) (shedding for ≤2 months) isolates from cattle and 16 bovine STEC isolates with unknown colonization types. Genoserotypes and multilocus sequence types (MLSTs) were determined, and the isolates were probed with a DNA microarray for virulence-associated genes (VAGs). All STEC(per) isolates belonged to only four genoserotypes (O26:H11, O156:H25, O165:H25, O182:H25), which formed three genetic clusters (ST21/396/1705, ST300/688, ST119). In contrast, STEC(spo) isolates were scattered among 28 genoserotypes and 30 MLSTs, with O157:H7 (ST11) and O6:H49 (ST1079) being the most prevalent. The microarray analysis identified 139 unique gene patterns that clustered with the genoserotypes and MLSTs of the strains. While the STEC(per) isolates possessed heterogeneous phylogenetic backgrounds, the accessory genome clustered these isolates together, separating them from the STEC(spo) isolates. Given the vast genetic heterogeneity of bovine STEC strains, defining the genetic patterns distinguishing STEC(per) from STEC(spo) isolates will facilitate the targeted design of new intervention strategies to counteract these zoonotic pathogens at the farm level., Importance: Ruminants, especially cattle, are sources of food-borne infections by Shiga toxin-producing Escherichia coli (STEC) in humans. Some STEC strains persist in cattle for longer periods of time, while others are detected only sporadically. Persisting strains can serve as gene reservoirs that supply E. coli with virulence factors, thereby generating new outbreak strains. Attempts to reduce the human risk for acquiring STEC infections should therefore include strategies to control such persisting STEC strains. By analyzing representative genes of their core and accessory genomes, we show that bovine STEC with a persistent colonization type emerged independently from sporadically colonizing isolates and evolved in parallel evolutionary branches. However, persistent colonizing strains share similar sets of accessory genes. Defining the genetic patterns that distinguish persistent from sporadically colonizing STEC isolates will facilitate the targeted design of new intervention strategies to counteract these zoonotic pathogens at the farm level., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

The in vitro effect of cyclosporin A (CsA) on lipid peroxidation in human liver microsomes was investigated, and efforts were made to prevent the resulting toxic effect of CsA. Microsomes were prepared from human liver resection material and incubated with CsA (0, 10, 30, 100, 300, 1000 micrograms/mL) for one hour (pH 7.4, 37 degrees, 95% O2, 5% CO2). Subsequently the resulting concentrations of malondialdehyde equivalents (MDA) were determined, a breakdown product of lipid peroxidation. Furthermore the duration of incubation was varied (0, 15, 30, 60, 90 min) using a CsA concentration of 300 micrograms/mL. CsA was shown to stimulate MDA-formation to up to 10-fold of the control value in both a time and concentration dependent manner. The dosage dependent experiment stated above was repeated, adding alpha-tocopherol (vitamin E, 1 mM), reduced glutathione (GSH, 1 mM), N-acetylcysteine (0.1, 0.3, 1, 3 mM), and Ginkgo biloba extract (Gbe, 15, 50, 150 micrograms/mL), respectively, to the medium of incubation. Vitamin E, a potent radical scavenger, proved to inhibit lipid peroxidation almost totally. Both GSH and N-acetylcysteine were also able to prevent lipid peroxidation, suggesting that the antioxidant effect of GSH might be caused by its thiol group and does not depend on the integrity of the whole molecule. Gbe inhibited CsA induced lipid peroxidation in a concentration dependent manner. This effect of Gbe was diminished yet not totally abolished when FeCl3 was added to the medium of incubation, whereas N-acetylcysteine even slightly enhanced CsA stimulated lipid peroxidation in the presence of iron. These results suggest that Gbe might be able to prevent radical mediated damage to human membranes caused by CsA.